Project Summary: The familial cancer syndrome, von Hippel-Lindau (VHL) disease, is caused by inherited mutations in the VHL tumor suppressor gene. Previous work has shown that VHL is a crucial regulatory gene in renal cells, controlling their differentiation and causing cell cycle and growth arrest specifically in cells that have been cultured previously at high cell density and subsequently grown on certain extracellular matrices. Additional VHL roles in protection of renal cells from apoptotic stimuli and in transport and deposition of fibronectin have been demonstrated. The most recognized biochemical function of VHL gene products (hereafter called pVHL) is as an integral member of an E3 ubiquitin ligase complex that targets specific substrates for ubiquitination and subsequent degradation. A large body of work has focused on one such pVHL substrate, hypoxia-inducible factor alpha (HIF-a) subunits. Inactivation of VHL results in high levels of HIF-a, leading to up-regulation of hypoxia-inducible genes at normal oxygen levels. However, it is unclear which cell biological functions of pVHL, especially those described above, are dependent on the ubiquitination of HIF-a and down-regulation of hypoxia-inducible genes. Notably, VHL mutations which do not impair the ability to ubiquitinate HIF-a have been detected in VHL-associated tumors, supporting the hypothesis that pVHL must have additional cellular functions. This project will explore these pVHL functions in renal cells and other cell types in order to clarify their relationship with HIF-a regulation. The specific aims of this proposal are: 1) To determine the contribution of HIF-a regulation toward VHL-associated cellular phenotypes and 2) To identify and examine alternative functions of pVHL through analysis of VHL mutations and associated cellular phenotypes. The proposed investigations will utilize established cell culture systems, with genetic manipulation of HIF-a levels and/or expression of mutant VHL proteins. The overall goal is to determine the underlying cellular mechanisms responsible for the observed VHL-associated phenotypic changes and for tumorigenesis in VHL disease. It is anticipated that these studies will also provide insight into cellular differentiation and growth arrest mechanisms applicable to a wider range of tumor types and foster a better understanding of tumorigenic processes at the cellular level. Relevance: Disruptions of the VHL tumor suppression gene lead to kidney, adrenal, and blood vessel tumors. The proposed research is aimed at understanding the changes that occur to a cell as a result of VHL disruption, and how these changes may result in tumor formation. [unreadable] [unreadable] [unreadable] [unreadable]